1. Field of the Invention
The present invention relates to a composition containing polyethylene glycol (PEG) and methanol for preservation of a cell or tissue, especially at ambient temperature. It may also be used for cell or tissue storage. A cell or tissue preserved with and/or stored in compositions of the present invention maintains its morphological characteristics, the recognition of its antigens by cognate antibodies, and the integrity of its nucleic acids (e.g., DNA and RNA) without requiring refrigeration or freezing.
2. Description of the Related Art
Cytological and histological processing prevents autolysis of cells and tissue, respectively, after their removal from a living body. Moreover, the structure of individual cells and their organization within the tissue are stabilized by such processing. There is a requirement, however, for sophisticated procedures and dedicated instruments in most cases to process cells and tissues in a clinical setting. Therefore, specimens are usually collected in physician offices or surgical suites, and transported to a centralized pathology service. Suitable compositions for the preservation and/or storage of a cell or tissue are needed to ensure that autolysis is prevented and that cellular morphology, antigen, and nucleic acid are maintained until processing.
Furthermore, genetic analysis is becoming more important by itself or complementary to cell staining, enzyme assays, and immunological techniques in pathology. Expression of mutant genes or the over-expression of normal genes can be examined by analyzing nucleic acid. In situ detection of RNA can localize transcripts within tissue containing different types of cells; this can also be accomplished by detecting RNA that has been extracted from different portions of sorted cells or sectioned tissue. Mutations may be seen in DNA or RNA. Alternating cytologic/histologic and genetic analyses of sorted cells or sectioned tissue can be used to correlate pathological events at cellular and molecular levels. Genetic analysis will be possible only if degradation is prevented and macromolecular structures are stabilized. But many preservative compositions and fixatives cause irreversible damage (e.g., activity of the ubiquitous nuclease enzymes, hydrolysis of phosphodiester bonds, and/or deamidation of bases) to the structure of nucleic acids (e.g., DNA, and especially RNA) and reduce their yield, thereby limiting the usefulness of genetic techniques for diagnosis and research applications. Consequently, preservation of nucleic acids in a fresh cell or tissue usually requires special handling, such as immediate processing or freezing, to allow examination by a combination of cytologic, histologic, immunologic, and genetic techniques.
The composition disclosed herein may be used to advantage in conventional tissue processing or other processing methods such as described in U.S. Pat. No. 6,207,408; WO 01/44783; and WO 01/44784. Conventional techniques are described in general references such as Thompson (Selected Histochemical and Histopathological Methods, Springfield, Ill.: Thomas, 1966), Sheehan & Hrapchak (Theory and Practice of Histotechnology, St. Louis, Mo.: Mosby, 1973), Bancroft & Stevens (Theory and Practice of Histological Techniques, New York, N.Y.: Churchill Livingstone, 1982); Boon & Kok (Microwave Cookbook of Pathology, Leiden, NL: Coulomb, 1989); Woods & Ellis (Laboratory Histopathology, New York, N.Y.: Churchill Livingstone, 1994).
U.S. Pat. Nos. 3,389,052; 3,546,334; 5,104,640; 5,256,571; 5,849,517; and 6,204,375; Florell et al. (Mod. Pathol., 14:116–128, 2001); Bostwick et al. (Arch. Pathol. Lab. Med., 118:298–302, 1994); Dimulescu et al. (Mol. Diagnosis, 3:67–71, 1998); Maxwell et al. (J. Clin. Pathol., 52:141–144, 1999) Shibutani et al. (Lab. Invest., 80:199–208, 2000); and Gillespie et al. (Am. J. Pathol., 160:449–457, 2002) describe preservative and fixative solutions.
Compositions of the present invention are novel and nonobvious. They are nonaqueous solutions comprising PEG and methanol, which preserve morphological characteristics, recognition of antigen by cognate antibody, and integrity of nucleic acid (e.g., DNA and RNA) in an isolated cell or solid tissue without the inconvenience of cooling or freezing the specimen to prevent degradation. Thus, an isolated cell or solid tissue can be stored for long times at ambient temperature. Further advantages of and improvements due to the invention are discussed below.